Ortho-polychlorinated biphenyls (o-PCBs, non-dioxin-like PCBs) are abundant Superfund chemicals that have a broad range of adverse effects linked to developmental exposure in vertebrates, including altered cognitive and behavioral outcomes. The proposed studies will explore mechanisms of o-PCB effects in the biological model zebrafish (Danio rerio) and the ecological model Atlantic killifish (Fundulus heteroclitus), to provide a mechanistic foundation for predicting and developing reliable markers of adverse outcomes linked to o-PCBs. O-PCBs are orders of magnitude more abundant than dioxin-like non-ortho substituted PCBs in the environment, in humans and in wildlife, increasing the concerns for significant health and ecological effects. PCB levels in killifish from the Superfund site in New Bedford Harbor, MA (NBH) are an extreme example, being as much as a thousand times greater than levels in other populations. Over generations of exposure, NBH killifish have become tolerant to dioxin-like PCBs, and our studies now suggest, to o-PCBs, as well. Our data show that the ortho-congener PCB153 causes large changes in gene expression in zebrafish and killifish larvae exposed as embryos, and in adult killifish brain, suggesting that there are heretofore unrecognized pathways and genes involved in genetic and behavioral responses to o-PCBs. We will examine response mechanisms in the brain and establish the contribution of nuclear receptor transcription factors and target genes to phenotypic and behavioral outcomes in exposed fish. Our recent data indicate that effects of different ortho-PCB congeners may involve a number of nuclear receptors. The o-PCB congener PCB153 misregulates large numbers of genes in various physiological pathways in developing zebrafish and killifish, and in adult killifish forebrain. We will expose adult and larval zebrafish and killifish from NBH and a reference site (Scorton Creek, MA) to o-PCB congeners, and use transcriptomics and proteomics to determine receptor-driven networks in brain. Interaction of o-PCB congeners with candidate zebrafish and killifish nuclear receptors, PXR (a major ?xenobiotic receptor?), PPARs, FXR, and LXR will be determined in vitro using reporter gene assays; in silico by modeling receptor proteins and docking o-PCBs; and in vivo using receptor knockout zebrafish generated by CRISPR-cas9 technology. The roles of receptors and target genes in behavioral outcomes will be examined using loss-of-function approaches in zebrafish and in killifish. Altered gene regulation and behavioral effects will be examined also in adults raised from exposed embryos, to determine whether early exposure produces lasting effects. Ecologically relevant effects may be enhanced or muted in response to chemical exposure in the New Bedford Harbor PCB-adapted fish population, or in other fishes exposed in the wild. The studies will provide new, fundamental information and tools important for risk assessment of established toxicants and related emerging chemicals of concern, which will facilitate ecologically relevant assessment of aquatic Superfund sites, including the success of remediation.